Sequential solid-state fermentation and enzymatic hydrolysis of green seaweed to extract antioxidant compounds

Nowadays, the seaweed industries are focused on the production of a single product, so they face the challenge of changing their strategies to generate multiple products from macroalgae biomass, following biorefinery concept. On the other hand, the on-site production of enzymes and sequential enzymatic hydrolysis, integrated to the biorefinery plant, is being considered as a potential strategy to avoid additional steps and reduce costs [1]. Solid-state fermentation (SSF) by filamentous fungi can produce carbohydrolases and sequentially hydrolyzes the biomass to sugars and antioxidant compounds [2]. In this form, extraction step of enzymes after SSF is avoided. In this study, it was carried out a sequential SSF and enzymatic hydrolysis of Ulva rigida (green seaweed). SSF was performed by Aspergillus ibericus with 10 g of dry seaweed with a moisture of 75%w/w (wet basis) at 25 ºC during 5 days. After SSF, a citrate buffer was added to carried out the enzymatic hydrolysis during 72h. This second step was optimized by Box-Behnken experimental design, in which the independent variables studied were the temperature (35, 40 and 45 ºC), the load of solid (10%, 20% and 30 % w/v) and pH (4, 4.6 and 5.2). The dependent variables studied were the variation of antioxidant activity and total phenolic compounds released. The model had a good fit for antioxidant activity (R2: 0.9947 and R2 adjusted: 0.9850) and for phenolic compounds (R2: 0.9528 and R2 adjusted: 0.8679). The independent variables that have a higher significant effect (p < 0.05) on the increase of antioxidant activity were load of solid and pH, and the interaction of these variables had the highest effect. Intermediate or low pH and high load of solid favored the released of antioxidant compounds. The model predicted an optimum value of antioxidant activity of 344 M Trolox equivalents at 41 ºC, 30 % w/v of solid and pH 4.7. For total phenolic compounds, the variable that had a higher significant effect (p < 0.05) was the temperature, the optimum conditions were 39 ºC, 30 % w/v of solid and pH 4. The bioprocessing of seaweed by SSF and enzymatic hydrolysis allowed to extract antioxidant compounds by environmentally friendly processes without the use of organic solvents and chemical or physical pre-treatments.Funded by the European Regional Development Fund (ERDF) under the project SPO3 (POCI-01-0145-FEDER-030377) and by Programa Operacional Mar2020, Portugal 2020 under the project InovFeed (ref. MAR-02.01.01-FEAMP-0111). Funded by Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004).info:eu-repo/semantics/publishedVersio

Bio-enrichment of oilseed cakes by Mortierella alpina under solid-state fermentation

"Available online 3 August 2020"Oilseed cakes have potential for new applications as substrates for solid-state fermentation (SSF), to increase their nutritional value by increasing its polyunsaturated fatty acids (PUFAs) or protein content. In this sense, it was performed a screening of oilseed cakes to be used as substrates for the production of PUFAs by Mortierella alpina Peyronel MUM 9412. Of all by-products tested, linseed cake (LSC) was the oilseed cake that achieved the highest production of total PUFAs: 153.09 ± 2.25 mg/g. Overall, the PUFAs and protein contents of fermented LSC increased 33% and 11%, respectively. Further, supplementation of the rapeseed cake with linseed oil, prior to the SSF, proved to increase the PUFAs production in about 26%. This study demonstrated the potential of SSF for improving linseed and linseed cakes nutritional composition and the positive effect of linseed oil as inductor to improve the PUFAs production by M. alpina.José Manuel Salgado was supported by grant CEB/N2020 – INV/01/ 2016 from Project “BIOTECNORTE - Underpinning Biotechnology to foster the north of Portugal bioeconomy” (NORTE-01-0145-FEDER000004). This work was supported by the project “Development of innovative sustainable protein and omega-3 rich feedstuffs for aquafeeds, from local agro-industrial by-products”, reference POCI-01-0145- FEDER-030377, funded by European Regional Development Fund (ERDF). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01- 0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Valorization of brewers spent grain using biological treatments and its application in feeds for European seabass (Dicentrarchus labrax)

Brewers spent grain (BSG) is the main brewery industry by-product, with potential applications in the feed and food industries due to its carbohydrate composition. In addition, the lignocellulosic nature of BSG makes it an adequate substrate for carbohydrases production. In this work, solid-state fermentation (SSF) of BSG was performed with Aspergillus ibericus, a non-mycotoxin producer fungus with a high capacity to hydrolyze the lignocellulosic matrix of the agro-industrial by-products. SSF was performed at different scales to produce a crude extract rich in cellulase and xylanase. The potential of the crude extract was tested in two different applications: -(1) - the enzymatic hydrolysis of the fermented BSG and (2) - as a supplement in aquafeeds. SSF of BSG increased the protein content from 25% to 29% (w/w), while the fiber content was reduced to 43%, and cellulose and hemicellulose contents were markedly reduced to around 15%. The scale-up of SSF from 10 g of dry BSG in flasks to 50 g or 400 g in tray-type bioreactors increased 55% and 25% production of cellulase and xylanase, up to 323 and 1073 U g1 BSG, respectively. The optimum temperature and pH of maximal activities were found to be 55°C and pH 4.4 for xylanase and 50°C and pH 3.9 for cellulase, cellulase being more thermostable than xylanase when exposed at temperatures from 45°C to 60°C. A BoxBehnken factorial design was applied to optimize the hydrolysis of the fermented BSG by crude extract. The crude extract load was a significant factor in sugars release, highlighting the role of hydrolytic enzymes, while the load of fermented BSG, and addition of a commercial -glucosidase were responsible for the highest phenolic compounds and antioxidant activity release. The lyophilized crude extract (12,400 and 1050 U g1 lyophilized extract of xylanase and cellulase, respectively) was also tested as an enzyme supplement in aquafeed for European seabass (Dicentrarchus labrax) juveniles. The dietary supplementation with the crude extract significantly improved feed and protein utilization. The processing of BSG using biological treatments, such as SSF with A. ibericus, led to the production of a nutritionally enriched BSG and a crude extract with highly efficient carbohydrases capable of hydrolyzing lignocellulosic substrates, such as BSG, and with the potential to be used as feed enzymes with remarkable results in improving feed utilization of an important aquaculture fish species.This study was supported by the project “SPO3-Development of innovative sustainable protein and omega-3 rich feedstuffs for aquafeeds, from local agro-industrial by-products”, reference POCI-01-0145-FEDER-030377, funded by the European Regional Development Fund (ERDF) and Portuguese Foundation for Science and Technology (FCT) and by the strategic funding of the UIDB/04469/2020 unit. HF and CC were supported by grants SFRH/BD/131219/2017 and SFRH/BPD/114942/2016, respectively, from FCT, MCTES, FSE, and UE under the North Portugal Regional Operational Program (NORTE 2020). José Manuel Salgado was supported by the grant CEB/N2020—INV/01/2016 from Project “BIOTECNORTE—Underpinning Biotechnology to foster the north of Portugal bioeconomy” (NORTE-01-0145-FEDER-000004).info:eu-repo/semantics/publishedVersio

Production of lipids and polyunsaturated fatty acids by solid-state fermentation

Dissertação de mestrado em BiotecnologiaSolid-state fermentation (SSF) is a promising biotechnological process that allows the use of inert supports to the growth of microorganisms to obtain value-added products such as lipids. The use of this type of supports for the realization of SSF, such as polyurethane foam (PUF), offers better extraction processes and allows this residual plastic material to be reused. Polyunsaturated fatty acids (PUFAs) are essential in healthy diets and their production is extremely important, since mammals cannot synthesize them, so they need to ingest them from food sources. They are present in animal products such as marine fish oil, however there are associated problems such as overfishing of the oceans. Thus, microbial oil is a preferable source of PUFAs. In addition, other microbial lipids of high industrial interest can be produced using SSF in an inert support, such as stearic, oleic and palmitic acid. For this reason, the lipid production in SSF was evaluated using two microorganisms: a filamentous fungus (Mortierella alpina Peyronel 9412) and a yeast (Yarrowia lipolytica W29). In a first study, the culture medium for the production of PUFAs by M. alpina Peyronel 9412 in SSF was optimized using an inert support (PUF). The highest amount of total PUFAs (535.41 ± 24.12 mg/g PUF), linoleic acid (129.66 ± 5.84 mg/g PUF) and α-linoleic acid (401.93 ± 18.10 mg/g PUF) were produced when the culture medium contained 20 g/L glucose, 10% (w/v) linseed oil, the C/N ratio was adjusted to 25 and the incubation temperature was 25 °C for 3 days decreasing to 16 °C on the remaining 4 days of fermentation. In addition, a hemicellulosic hydrolysate absorbed to PUF was also used as a low-cost culture medium in SSF, with the growth and production of PUFAs being observed by the fungus, although in a smaller amount than with the synthetic medium. On the other hand, in a second study was tested the growth of yeast Y. lipolytica W29 and the production of lipids in SSF using PUF as well as its reuse in several fermentation cycles. It was found that the highest amount of lipids was obtained on the sixth day of fermentation. The fatty acid profile produced by this yeast showed a large amount of stearic acid (31.77 ± 1.59%), followed by palmitic acid (30.05 ± 1.50%) and linoleic acid (19.52 ± 0.98%). It was also possible to verify that when the PUF was squeezed, the cells were released to the culture medium, therefore, it is not useful to extract lipids from the support, since the biomass that is attached to it is reduced. Finally, 3 cycles of fermentation of 6 days each were carried out and only inoculum was added at the beginning of the first cycle. It was verified that when the PUF is squeezed after each fermentation cycle, some cells remain attached to it, being able to multiply with the addition of fresh medium. Through this study it was possible to conclude that SSF using an inert support such as PUF is a suitable biotechnological process for the production of value-added compounds such as lipids and, more specifically, PUFAs. The optimization of parameters such as temperature, C/N ratio and the addition of inductors will allow to define the fermentation conditions of other natural substrates such as agroindustrial residues.A fermentação em estado sólido (SSF) é um processo biotecnológico promissor que permite o uso de suportes inertes para o crescimento de microrganismos de forma a obter produtos de valor acrescentado como lípidos. O uso deste tipo de suportes para a realização de SSF, como espuma de poliuretano (PUF), oferece melhores processos de extração e permite que este material plástico residual seja reaproveitado. Os ácidos gordos polinsaturados (PUFAs) são essenciais em dietas saudáveis e a sua produção é extremamente importante, uma vez que os mamíferos não conseguem sintetizalos, por isso precisam de ingeri-los de fontes alimentares. Existem em produtos de origem animal como o óleo de peixe marinho, no entanto existem problemas associados, como a sobrepesca dos oceanos. Assim, o óleo microbiano é uma fonte preferível de PUFAs. Além disso, outros lípidos microbianos com elevado interesse industrial podem ser produzidos utilizando SSF em suporte inerte, como é o caso do ácido esteárico, linoleico, oleico e palmítico. Por este motivo foi avaliada a produção de lípidos em SSF usando dois microrganismos: um fungo filamentoso (Mortierella alpina Peyronel 9412) e uma levedura (Yarrowia lipolytica W29). Num primeiro estudo foi otimizado o meio de cultura para a produção de PUFAs por M. alpina Peyronel 9412 em SSF utilizando um suporte inerte (PUF). Verificou-se que a maior quantidade de PUFAs totais (535.41 ± 24.12 mg/g PUF), ácido linoleico (129.66 ± 5.84 mg/g PUF) e ácido α-linoleico (401.93 ± 18.10 mg/g PUF) foi produzida quando o meio continha 20 g/L glucose, 10% (p/v) de óleo de linhaça, o rácio C/N era ajustado a 25 e a temperatura de incubação era de 25 ºC durante 3 dias descendo para 16 ºC nos restantes 4 dias de fermentação. Para além disso, foi também utilizado um hidrolisado hemicelulósico absorvido ao PUF, como meio de cultura de baixo custo em SSF, verificando-se o crescimento e produção de PUFAs por parte do fungo, ainda que em menor quantidade do que com o meio sintético. Por outro lado, num segundo estudo foi testado o crescimento da levedura Y. lipolytica W29 e a produção de lípidos em SSF utilizando PUF e a sua reutilização em vários ciclos de fermentação. Verificou-se que a maior quantidade de lípidos foi obtida ao sexto dia de fermentação. O perfil de ácidos gordos produzidos por esta levedura indica uma grande quantidade de ácido esteárico (31.77 ± 1.59%), seguido de ácido palmítico (30.05 ± 1.50%) e linoleico (19.52 ± 0.98%). Foi ainda possível verificar que quando a PUF foi espremida, as células saíram para o meio de cultura, não sendo, portanto, útil extrair lípidos do suporte, já que a biomassa que fica unida a ele é reduzida. Por fim, foram realizados 3 ciclos de fermentação de 6 dias cada um e só foi adicionado inóculo no início da primeira fermentação, verificando-se que, quando a PUF é espremida após cada ciclo de fermentação, algumas células permanecem unidas a ela, conseguindo multiplicar-se com a adição de meio fresco. Através deste estudo foi possível concluir que a SSF utilizando um suporte inerte como PUF é um processo biotecnológico adequado a produção de compostos de valor acrescentado como lípidos e, mais concretamente, PUFAs. A otimização dos parâmetros como a temperatura, ratio C/N e a adição de indutores permitirá definir as condições de fermentação de outros substratos naturais como os resíduos agroindustriais.The work related with polyunsaturated fatty acid production was supported by the R&D&I project “Development of innovative sustainable protein and omega-3 rich feedstuffs for aquafeeds, from local agro-industrial by-products”, reference POCI-01-0145-FEDER-030377, funded by European Regional Development Fund (ERDF). The work related to production of lipids by Y. lipolytica was supported by the R&D&I project “Lipid Production by Yarrowia lipolytica growing on waste/wastewater streams”, reference TUBITAK/0009/2014, funded by FCT - Fundação para a Ciência e a Tecnologia

Global economic burden of unmet surgical need for appendicitis

Background There is a substantial gap in provision of adequate surgical care in many low- and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially

Global economic burden of unmet surgical need for appendicitis

Background There is a substantial gap in provision of adequate surgical care in many low- and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially